This proposal focuses on identifying natural compounds and combinations of natural compounds that behave as calorie restriction (CR) mimetics. Ursolic acid (UA), a pentacyclic triterpene compound found in rosemary, apples, berries, Perilla frutescens (P. frutescens) and other sources, as well as several other related triterpenoids have been shown to inhibit skin tumor promotion by TPA and to block both Akt signaling as well as NFkB signaling in other studies. Evidence exists that UA may also modulate activity of the glucocorticoid receptor (GR). In preliminary studies, including new data added to this application, we have found that UA had inhibitory effects on epidermal Akt, NFkB, p38 MAPK and JNK activation induced by TPA treatment. UA also inhibited TPA-induced epidermal hyperproliferation. Notably, UA, when given in combination with resveratrol (Res), appeared to produce synergistic inhibition of these signaling pathways, as well as TPA-induced epidermal hyperproliferation. Furthermore, combinations of UA and Res dramatically induced activation of epidermal AMPK following treatment with TPA. Preliminary experiments suggest that UA modulates the GR in a human keratinocyte cell line. Additional new data demonstrates that Rapa, UA, Res and the combination of UA+RES suppress the proliferation of putative stem cells from the bulge region of hair follicles. We have made significant progress in the synthesis/preparation of additional pentacyclic triterpenes found in P. frutescens. We propose to use the well-characterized two-stage skin carcinogenesis model to explore the mechanisms that underlie the chemopreventive properties of UA and a series of related pentacyclic triterpenes. In addition, we will explore the possibility that UA (or a more potent triterpene found in P. frutescens) when combined with other phytochemicals such as Res, curcumin (Curc), 6-shogaol (6Sho) or Rapa will produce either additive or possibly synergistic chemopreventive effects. We will test the hypothesis that UA alone or in combination with other phytochemicals acts as a CR mimetic by modulating growth factor signaling, inflammatory signaling and possibly other signaling pathways to suppress proliferation of initiated keratinocytes. The hypothesis that combinations of triterpenoid compounds (such as UA) together with other chemo- preventive chemicals/phytochemicals will produce additive or synergistic CR mimetic activity will also be tested. The specific aims are: i) Examine the effect of UA and a series of related pentacyclic triterpenes on epidermal signaling pathways induced by TPA; ii) Examine the ability of UA and a series of related pentacyclic triterpenes to modulate epidermal GR function during tumor promotion; iii) Examine the effect of UA and a series of related triterpenes on keratinocyte proliferation (including bulge region KSCs) and skin inflammation in relation to their ability to inhibit skin tumor promotion by TPA; iv) Examine whether UA combined with other anti-inflammatory chemicals can achieve synergistic inhibitory effects toward skin tumor promotion by TPA. PUBLIC HEALTH RELEVANCE: Research in this proposal will evaluate the underlying mechanism(s) for the chemopreventive action of ursolic acid (UA) and a series of related pentacyclic triterpenes from P. frutescens that appear to possess calorie restriction (CR) mimetic properties. In addition, we will explore combinations of several different agents together with UA for possible additive or synergistic inhibitory effects on skin tumor promotion. The goal of these experiments will be to establish mechanisms for an important class of chemopreventive phytochemicals (pentacyclic triterpenes) and identify combinations of chemopreventive agents that could be used in humans to prevent the promotion stage of epithelial carcinogenesis in multiple tissues.